CN112852693B

CN112852693B - Recombinant escherichia coli for producing L-lactic acid and application thereof

Info

Publication number: CN112852693B
Application number: CN202011006212.2A
Authority: CN
Inventors: 张学礼; 刘萍萍; 唐金磊
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2020-09-17
Filing date: 2020-09-17
Publication date: 2021-11-16
Anticipated expiration: 2040-09-17
Also published as: CN112852693A

Abstract

The invention discloses recombinant escherichia coli for producing L-lactic acid and application thereof. The recombinant escherichia coli to be protected does not contain a D-lactate dehydrogenase gene and contains an L-lactate dehydrogenase mutant gene, the recombinant escherichia coli expresses the L-lactate dehydrogenase mutant gene, and an amino acid sequence of the L-lactate dehydrogenase mutant gene is a protein of a sequence 4 in a sequence table. After the recombinant escherichia coli is used for anaerobic fermentation for 48 hours, the yield of the L-lactic acid reaches 150g/L, the yield reaches 1.90mol/mol, and the optical purity approaches 100%, which shows that the recombinant escherichia coli constructed and obtained by the invention can produce the L-lactic acid with high yield and high optical purity, and can be applied to the production of high-quality L-lactic acid in the field of biological fermentation.

Description

Recombinant escherichia coli for producing L-lactic acid and application thereof

Technical Field

The invention relates to the field of industrial microorganisms, in particular to recombinant escherichia coli for producing L-lactic acid and application thereof.

Background

L-lactic acid is an important organic acid and widely used in the industries of food, medicine, tobacco, chemical industry and the like. In addition, with the increasing demand for green and environmentally friendly materials, the most important use of L-lactic acid in the future is as a monomer for synthesizing polylactic acid (PLA), which is a biodegradable material. Polylactic acid is a novel biodegradable material, and is widely applied to the industries of clothing, construction, agriculture, forestry and medicine due to excellent comprehensive performance. Meanwhile, polylactic acid is known as an environment-friendly material because it can be completely degraded by microorganisms in the nature to generate carbon dioxide and water after use. Therefore, as PLA is more widely used, the market potential of L-lactic acid in the future will be greater.

The lactic acid production mode mainly comprises a microbial fermentation method, a chemical synthesis method and an enzyme catalysis method. Among them, the enzyme catalysis method has not been used in industrial production because of the complex fermentation method. Chemical processes allow large-scale continuous production of lactic acid, and the synthesis of lactic acid has also been approved by the U.S. Food and Drug Administration (FDA), but its raw materials are mainly petroleum-based chemicals and are therefore greatly affected by crude oil supply and price fluctuations. In addition, chemical synthesis involves a large amount of toxic substances and produces lactic acid without optical specificity, and thus is relatively less applicable in industry. The method for producing lactic acid by using renewable raw materials, such as corn starch or cassava powder, as substrates and utilizing microorganisms through fermentation can efficiently synthesize the lactic acid with specific optical characteristics. Lactic acid produced by the current fermentation method accounts for more than 70 percent of the global lactic acid production.

There are many bacteria naturally producing lactic acid in nature, and these bacteria are abundant in various species and widely distributed in nature, such as lactobacillus, bacillus, bifidobacterium, lactococcus, etc. The bacteria have strong acid resistance, and can realize homotype fermentation production of L-lactic acid after genetic modification. However, these bacterial fermentation processes require rich media and cannot utilize pentoses, which greatly increases the production cost and the cost of downstream separation and purification. In addition, such bacteria typically use calcium hydroxide or calcium carbonate as a neutralizing agent to control the fermentation pH, and the final fermentation product is calcium lactate. Calcium lactate usually needs to be added with sulfuric acid in the separation and extraction process, and a large amount of calcium sulfate waste is generated and is difficult to treat. More importantly, the chiral purity of the L-lactic acid produced by the bacteria is below 98 percent, and the requirement of polylactic acid production cannot be met.

With the development of synthetic biology and metabolic engineering, the engineering bacteria obtained through rational design and genetic modification can obviously improve the performance indexes of lactic acid production in the aspects of L-lactic acid chirality, engineering bacteria substrate utilization range, strain tolerance and the like. Therefore, the engineering strain which can produce the L-lactic acid with high optical purity at low cost and high conversion rate and is used for producing the L-lactic acid with high quality still has strong necessity in the field of biological fermentation by genome-wide metabolic breeding and designing and constructing.

Disclosure of Invention

The technical problem to be solved by the invention is how to produce high-optical-purity and high-quality L-lactic acid in high yield or how to obtain an engineering strain for producing high-optical-purity L-lactic acid in high conversion rate.

In order to solve the above technical problems, the present invention first provides a recombinant E.coli.

The recombinant escherichia coli does not contain a D-lactate dehydrogenase gene and contains an L-lactate dehydrogenase mutant gene, and the amino acid sequence of the L-lactate dehydrogenase mutant gene is a protein shown in a sequence 4 in a sequence table.

The L-lactate dehydrogenase mutant gene is any one of the following DNA molecules:

c1, the coding sequence of the coding chain is DNA or cDNA molecule of 89-1051 bit nucleotide of sequence 3 in the sequence table;

c2, a DNA or cDNA molecule which hybridizes under stringent conditions with the DNA molecule defined by c1 and encodes the L-lactate dehydrogenase mutant;

c3, a DNA or cDNA molecule having more than 80% identity with the DNA molecule defined by c1 or c2 and encoding the L-lactate dehydrogenase mutant.

The stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: in a solution of 6 XSSC, 0.5% SDS at 65 ℃ and then washed once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

The above "identity" refers to sequence similarity to a native nucleic acid sequence. "identity" can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The L-lactate dehydrogenase mutant gene is ldhL, and the encoded polypeptide contains a modification of replacing T with A at the 45 th position and a modification of replacing G with S at the 258 th position in the amino acid sequence shown in a sequence 4 in a sequence table; optionally, the recombinant escherichia coli has increased expression of ldhL, and/or increased activity of the protein encoded by ldhL.

The recombinant escherichia coli is obtained by modifying a receptor escherichia coli, wherein the receptor escherichia coli is escherichia coli Dlac-206, and the registration accession number of the escherichia coli Dlac-206 in the China general microbiological culture Collection center is CGMCC No. 7679.

The recombinant escherichia coli contains an L-lactate dehydrogenase mutant gene expression cassette, and the L-lactate dehydrogenase mutant gene expression cassette contains a promoter and the L-lactate dehydrogenase mutant gene driven by the promoter.

The promoter is M1-93, and the M1-93 promoter is any one of the following DNA molecules:

1) the nucleotide sequence of one strand is DNA molecule of 1 st-88 th nucleotide of sequence 3 in the sequence table;

2) a DNA molecule having an identity of 80% or more to the DNA molecule of 1) and having a promoter function.

The L-lactate dehydrogenase mutant gene expression cassette can be a double-stranded DNA molecule with a chain of nucleotides with the nucleotide sequence of 1 st to 1051 th of a sequence 3 in a sequence table.

The L-lactate dehydrogenase mutant gene expression cassette is positioned at the coding gene (frd for short) site of the fumarate reductase of the receptor escherichia coli.

The recombinant Escherichia coli can be obtained by modifying Escherichia coli Dlac-206 as follows: the D-lactate dehydrogenase encoding gene (abbreviated as ldhA) of Escherichia coli Dlac-206 was deleted, and an L-lactate dehydrogenase mutant gene expression cassette was introduced into the site of the fumarate reductase encoding gene (abbreviated as frd) of Escherichia coli Dlac-206. The nucleotide sequence of the L-lactate dehydrogenase mutant gene expression cassette is sequence 3 in the sequence table. The sequence 3 in the sequence table is composed of 1051 nucleotides, the 1 st to 88 th nucleotides are M1-93 promoters, and the 89 th to 1051 th nucleotides are CDS of L-lactate dehydrogenase mutant genes.

Among them, the deletion of the D-lactate dehydrogenase-encoding gene (ldhA for short) of E.coli Dlac-206 can be achieved by homologous recombination. Introduction of the L-lactate dehydrogenase mutant gene expression cassette into the site of the fumarate reductase coding gene (frd for short) of E.coli Dlac-206 can be achieved by homologous recombination.

The recombinant escherichia coli is recombinant escherichia coli Slac007 which has a registration accession number of CGMCC No.19459 in the China general microbiological culture Collection center.

In order to solve the above technical problems, the present invention further provides a method for constructing the recombinant escherichia coli described above, comprising transforming the recipient escherichia coli as follows to obtain the recombinant escherichia coli: knocking out the D-lactate dehydrogenase gene of the receptor escherichia coli, and expressing the L-lactate dehydrogenase mutant gene in the receptor escherichia coli.

The construction method specifically comprises the steps of knocking out a D-lactate dehydrogenase encoding gene (abbreviated as ldhA) of escherichia coli Dlac-206, and introducing the L-lactate dehydrogenase mutant gene expression cassette into a fumarate reductase encoding gene (abbreviated as frd) site of the escherichia coli Dlac-206. The nucleotide sequence of the L-lactate dehydrogenase mutant gene expression cassette is sequence 3 in the sequence table. The sequence 3 in the sequence table is composed of 1051 nucleotides, the 1 st to 88 th nucleotides are M1-93 promoters, and the 89 th to 1051 th nucleotides are CDS of L-lactate dehydrogenase mutant genes.

In order to solve the above technical problems, the present invention also provides a method for producing L-lactic acid, comprising culturing any of the above recombinant E.coli to obtain a fermentation product; obtaining L-lactic acid from the fermentation product. In the method, ammonia water is used as a neutralizer to adjust the pH value in the culture process.

The invention also provides a protein or a biological material related to the protein, which is characterized in that: the protein is a protein with an amino acid sequence shown in a sequence 4 in a sequence table, and the biological material is any one of the following B1) to B9):

B1) a nucleic acid molecule encoding the protein;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic cell line comprising B1) the nucleic acid molecule or a transgenic cell line comprising B2) the expression cassette;

B6) transgenic tissue containing the nucleic acid molecule of B1) or transgenic tissue containing the expression cassette of B2); B7) a transgenic organ containing the nucleic acid molecule of B1), or a transgenic organ containing the expression cassette of B2); B8) a nucleic acid molecule that inhibits or reduces the expression of the protein in the above-described applications;

B9) an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line comprising the nucleic acid molecule according to B8).

In the above biological material, the nucleic acid molecule of B1) may be a gene encoding the protein as shown in B1) B2) or B3):

b1) the coding sequence (ORF) of the coding strand is a DNA molecule of 89 th to 1051 th nucleotides of a sequence 3 in a sequence table;

b2) the nucleotide sequence of the coding chain is a DNA molecule of a sequence 3 in a sequence table;

b3) a DNA molecule which hybridizes with the DNA molecule defined in 2) under stringent conditions and encodes a protein having the same function.

The above stringent conditions are hybridization and washing of the membrane 2 times 5min at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and hybridization and washing of the membrane 2 times 15min at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

Any of the following applications also fall within the scope of the present invention:

the application of P1, the recombinant Escherichia coli described above or the construction method of the recombinant Escherichia coli described above in the production of L-lactic acid,

the use of P2, a protein as described above or a biological material related thereto for the production of L-lactic acid,

the application of P3, the recombinant escherichia coli described above or the construction method of the recombinant escherichia coli described above in the preparation of L-lactate dehydrogenase;

the application of P4, the L-lactate dehydrogenase mutant gene or the L-lactate dehydrogenase mutant gene expression cassette in constructing recombinant Escherichia coli for producing L-lactate dehydrogenase;

the use of P5, the L-lactate dehydrogenase mutant gene described above, or the L-lactate dehydrogenase mutant gene expression cassette described above for the preparation of L-lactate dehydrogenase;

use of P6, a protein as described above or a biological material related thereto for the preparation of an L-lactate dehydrogenase preparation.

The invention constructs an engineered recombinant escherichia coli for producing L-lactic acid, the strain number is Slac007, and the registration number of the engineered recombinant escherichia coli in the China general microbiological culture Collection center is CGMCC No. 19459. After the recombinant escherichia coli is used for anaerobic fermentation for 48 hours, the yield of the L-lactic acid reaches 150g/L, the yield reaches 1.90mol/mol, and the optical purity approaches 100%, which shows that the recombinant escherichia coli constructed and obtained by the invention can produce the L-lactic acid with high yield and high optical purity, and can be applied to the production of high-quality L-lactic acid in the field of biological fermentation.

Deposit description

The strain name is as follows: escherichia coli

Latin name: escherichia coli

The strain number is as follows: slac007

The preservation organization: china general microbiological culture Collection center

The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)

Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district

The preservation date is as follows: 3 and 6 months in 2020

Registration number of the preservation center: CGMCC No.19459

Drawings

FIG. 1 shows OD of fermentation broth obtained by fermenting Escherichia coli Slac006 at different times_550nmGlucose and L-lactic acid.

FIG. 2 shows the culture temperature and OD of fermentation broth in the construction of recombinant Escherichia coli Slac007_550nm。

FIG. 3 is an HPLC chromatogram of a fermentation broth obtained from a 500mL fermentor of a standard and recombinant Escherichia coli Slac 007. The upper graph is HPLC spectra of a D-lactic acid standard substance and an L-lactic acid standard substance; the retention time of the D-lactic acid standard was 8.189 minutes; the retention time of the L-lactic acid standard was 9.035 minutes. The lower graph is an HPLC (high performance liquid chromatography) spectrum of fermentation liquor obtained by recombinant Escherichia coli Slac007 in a 500mL fermentation tank; the retention time of the L-lactic acid standard was 9.055 minutes.

FIG. 4 shows OD of fermentation broth obtained by fermenting Slac007 in 5L tank for different time periods₅₅₀nm, glucose and L-lactic acid content.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Escherichia coli Dlac-206 in the examples described below was deposited in the China general microbiological culture Collection center (CGMCC) at 04.6.2013, and the accession number thereof was CGMCC No. 7679. Chinese patent publication No. CN 104278003B, which has been published on 18.12.2018 (see paragraph 0212 of the specification and claim 8). CN 104278003B discloses that after 48 hours of fermentation of E.coli Dlac-206, the lactic acid yield reaches 1133mM (equivalent to 102g/L), the yield reaches 1.94mol/mol (equivalent to 0.97g/g), and the optical purity of D-lactic acid is > 99.5% (see 0209 paragraph and 0030 paragraph of the specification).

In the following examples, Escherichia coli Dlac-206 was used as a receptor, and the engineered strain for efficient production of L-lactic acid with high optical purity, i.e., recombinant Escherichia coli Slac007, was obtained by the following transformation: the D-lactate dehydrogenase encoding gene (abbreviated as ldhA) of Escherichia coli Dlac-206 was deleted, and an L-lactate dehydrogenase mutant gene expression cassette was introduced into the site of the fumarate reductase encoding gene (abbreviated as frd) of Escherichia coli Dlac-206. The nucleotide sequence of the L-lactate dehydrogenase mutant gene expression cassette is sequence 3 in the sequence table. The sequence 3 in the sequence table is composed of 1051 nucleotides, the 1 st to 88 th nucleotides are M1-93 promoters, and the 89 th to 1051 th nucleotides are CDS sequences of L-lactate dehydrogenase mutant genes. Among them, the deletion of the D-lactate dehydrogenase-encoding gene (ldhA for short) of E.coli Dlac-206 can be achieved by homologous recombination. Introduction of the L-lactate dehydrogenase mutant gene expression cassette into the site of the fumarate reductase coding gene (frd for short) of E.coli Dlac-206 can also be achieved by homologous recombination.

Example 1 integration of the L-lactate dehydrogenase-encoding Gene into E.coli Dlac-206

The method is characterized in that a L-lactate dehydrogenase gene (ldhL for short) is integrated to a fumarate reductase coding gene (frd for short) site of escherichia coli Dlac-206 by a homologous recombination method to obtain recombinant engineering bacteria Slac002, and specifically comprises the following steps:

(1) extraction of Lactobacillus plantarum ATCC 14917 genomic DNA

Various microorganisms in nature can directly synthesize L-lactic acid. In order to achieve efficient L-lactate production in E.coli, the coding for L-lactate dehydrogenase from Lactobacillus plantarum (Lactobacillus plantarum) was selected according to the activity of L-lactate dehydrogenase reported in the literatureThe gene ldhL is used for constructing recombinant engineering bacteria. The Lactobacillus plantarum ATCC 14917 is obtained from China General Microbiological Culture Collection Center (China General Microbiological Culture Collection Center, Ministry of Western No.1, North Cheng Yang district, Beijing, China) with the Collection number of CGMCC 1.2437. Extraction of genomic DNA of Lactobacillus plantarum ATCC 14917 Using DNA extraction kit from Promega: (

Genomic DNA Purification Kit, cat # A1120), and the specific steps are completed according to the Kit instructions. The extracted genomic DNA was stored at-20 ℃ for future use.

(2) Integrating ldhL into a fumaric reductase coding gene (frd for short) site in escherichia coli Dlac-206 by a two-step homologous recombination method, and the specific contents comprise:

in the first step, a DNA fragment I of 2719bp, which comprises a nucleotide sequence of 50bp on the Frd upstream homology arm, a nucleotide sequence of 50bp on the cat-sacB expression cassette and a nucleotide sequence of 50bp on the Frd downstream homology arm, is amplified by using pXZ-CS plasmid (Tan, et al, Appl Environ Microbiol,2013,79:4838-4844) DNA as a template and using primers Frd-CS-up/Frd-CS-down (consisting of Frd-CS-up and Frd-CS-down in Table 2) for the first step of homologous recombination.

The amplification system is as follows: new England Biolabs Phusion 5 Xbuffer 10U l, dNTP (10 mM each dNTP) 1U l, DNA template 20ng, primer (10U M) 2U l, Phusion High-Fidelity DNA polymerase (2.5U/. mu.l) 0.5U l, distilled water 33.5U l, total volume 50U l.

Amplification conditions were 98 ℃ pre-denaturation for 2 min (1 cycle); denaturation at 98 ℃ for 10 seconds, annealing at 56 ℃ for 10 seconds, and extension at 72 ℃ for 2 minutes (30 cycles); extension at 72 ℃ for 10 min (1 cycle).

The above DNA fragment I was used for the first homologous recombination: first, pKD46 plasmid (Datsenko and Wanner2000, Proc Natl Acad Sci USA 97: 6640-. The DNA fragment I was then electroporated into pKD 46/Dlac-206.

The electrotransfer conditions were: first, an electrotransformation competent cell of pKD46/Dlac-206 was prepared (Dower et al, 1988, Nucleic Acids Res 16: 6127-6145); 50 μ l of competent cells were placed on ice, 50ng of DNA fragment I was added, placed on ice for 2 minutes, and transferred to a 0.2cm Bio-Rad cuvette. A MicroPulser (Bio-Rad) electroporator was used with a shock parameter of 2.5 kv. After the electric shock, 1ml of LB medium was quickly transferred to a cuvette, and after 5 strokes, transferred to a test tube, and incubated at 75rpm for 2 hours at 30 ℃. 200 mu l of bacterial liquid is taken and coated on an LB plate containing ampicillin (the final concentration is 100 mu g/ml) and chloramphenicol (the final concentration is 34 mu g/ml), after overnight culture at 30 ℃, single colonies are selected for PCR verification, the used primers XZ-frd-up/XZ-frd-down are used, the correct colony amplification product is a 3440bp fragment (comprising 426bp upstream of the frd gene, 2619bp downstream of the cat-sacB expression cassette and 395bp downstream of the frd gene), and a correct single colony is selected and named as Escherichia coli Slac 001.

In the second step, with Lactobacillus plantarum ATCC 14917 genomic DNA as a template, 1063bp DNA fragment II (comprising 50bp of the upstream homology arm of the Frd gene, 963bp of the coding sequence of the ldhL gene and 50bp of the downstream homology arm of the Frd gene) was obtained by amplification using primers Frd-ldh14917-up/Frd-ldh14917-down (consisting of 50bp of the upstream homology arm of the Frd gene, and 50bp of the downstream homology arm of the Frd gene) for the second homologous recombination. The amplification conditions and system were as described in the first step of example 1 (2). The DNA fragment II was electroporated into Slac001 harboring pKD46 plasmid.

The electrotransfer conditions were: first, electroconversion competent cells of Slac001 harboring pKD46 plasmid were prepared (Dower et al, 1988, Nucleic Acids Res 16: 6127-6145); 50. mu.l of competent cells were placed on ice, 50ng of DNA fragment II was added, and the mixture was placed on ice for 2 minutes and transferred to a 0.2cm Bio-Rad cuvette. A MicroPulser (Bio-Rad) electroporator was used with a shock parameter of 2.5 kv. After the electric shock, 1ml of LB medium was quickly transferred to a cuvette, and after 5 strokes, transferred to a test tube, and incubated at 75rpm and 30 ℃ for 4 hours. The culture broth was transferred to LB liquid medium without sodium chloride containing 10% sucrose (50 ml of medium in a 250ml flask), and after 24 hours of culture, streaked on LB solid medium without sodium chloride containing 6% sucrose. Through PCR verification, the used primers are XZ-frd-up/XZ-frd-down (composed of XZ-frd-up and XZ-frd-down in Table 2), the correct colony amplification product is a 1784bp fragment (containing 426bp upstream of the frd gene, 963bp of the ldhL coding region sequence and 395bp downstream of the frd gene), a correct single colony is selected and named as Escherichia coli Slac002 (Table 1). Slac002 is a recombinant bacterium obtained by integrating an L-lactate dehydrogenase gene (the nucleotide sequence is a sequence 1 in a sequence table) into a fumaric acid reductase coding gene (frd for short) site of escherichia coli Dlac-206. The strains and plasmids constructed in this study are detailed in Table 1, and the primers used are detailed in Table 2.

TABLE 1 strains and plasmids used in the invention

TABLE 2 primers used in the present invention

Example 2 control of expression of L-lactate dehydrogenase-encoding Gene (ldhL for short) Using an Artificial regulatory element

The method for controlling the expression intensity of the ldhL gene by using the artificial regulatory element comprises the following steps:

in the first step, a pXZ-CS plasmid (Tan, et al, Appl Environ Microbiol,2013,79:4838-4844) DNA was used as a template, and a DNA fragment I of 2719bp (comprising a nucleotide sequence of 50bp upstream of the Frd gene, a cat-sacB expression cassette 2619bp and an initial 50bp of ldhL gene) was amplified using primers Frd-CS-up/14917-M93CS-down (consisting of Frd-CS-up and 14917-M93CS-down in Table 2) for the first step of homologous recombination. The amplification system and amplification conditions were as described in example 1(2) first step.

The DNA fragment I was used for the first homologous recombination: first, pKD46 plasmid (Datsenko and Wanner2000, Proc Natl Acad Sci USA 97: 6640-. The DNA fragment I was then electroporated into pKD46/Slac 002.

The electrotransfer conditions and procedures were identical to the first procedure described in example 1 for integration of the ldhL gene. Mu.l of the bacterial suspension was applied to LB plate containing ampicillin (final concentration: 100. mu.g/ml) and chloramphenicol (final concentration: 34. mu.g/ml), incubated overnight at 30 ℃ and single colonies were selected for PCR verification, verified by using primers XZ-frd-up/14917-ProYZ346-down (consisting of XZ-frd-up and 14917-ProYZ346-down in Table 2), and correct PCR products of 3391bp (including upstream homology arm 426bp of the frd gene, cat-sacB expression cassette 2619bp and initial 346bp of the ldhL gene) were selected and a correct single colony was designated as E.coli Slac 003.

In the second step, 188bp of DNA fragment II (50 bp of upstream homology arm of Frd gene, 88bp of promoter sequence M1-93 and 50bp of start of ldhL gene) was amplified using genomic DNA of M1-93(Lu, et al, Appl Microbiol Biotechnol,2012,93: 2455-2455) as a template and primers Frd-M93-up/14917-M93-down (consisting of Frd-M93-up and 14917-M93-down in Table 2). The DNA fragment II was used for the second homologous recombination. The DNA fragment II was electroporated into the strain Slac 003.

The electrotransfer conditions and procedures were identical to the second procedure described in example 1 for integration of the ldhL gene. Cloning was verified by colony PCR using XZ-frd-up/14917-ProYZ346-down (consisting of XZ-frd-up and 14917-ProYZ346-down in Table 2) as primers and 860bp (426 bp upstream homology arm of frd gene, 88bp promoter sequence of M1-93, and 346bp initiation of ldhL gene) as a correct colony amplification product, and a correct single colony was selected and named as E.coli Slac004 (Table 1). The Escherichia coli Slac004 contains M1-93 promoter with nucleotide sequence of 1 st-88 th nucleotides of sequence 2 in the sequence table.

Example 3 deletion of D-lactate dehydrogenase-encoding Gene (ldhA for short)

Knocking out a D-lactate dehydrogenase coding gene (ldhA for short) of escherichia coli Slac004 by using a two-step homologous recombination method so as to realize high-efficiency production of L-lactic acid by using an engineering strain, and specifically comprising the following steps:

in the first step, a DNA fragment I of 2719bp (50 bp for the upstream homology arm of ldhA gene, 2619bp for the cat-sacB expression cassette 2619bp and 50bp for the downstream homology arm of ldhA gene) was amplified using pXZ-CS plasmid (Tan, et al, Appl Environ Microbiol,2013,79:4838-4844) DNA as a template and primers ldhA-delCS-up/ldhA-delCS-down (consisting of ldhA-delCS-up and ldhA-delCS-down in Table 2) for the first step of homologous recombination. The amplification system and amplification conditions were as described in example 1(2) first step.

The DNA fragment I was used for the first homologous recombination: the plasmid pKD46 (Datsenko and Wanner2000, Proc Natl Acad Sci USA 97: 6640-. The DNA fragment I was then electroporated into pKD46/Slac 004. The electrotransfer conditions and procedures were identical to the first procedure described in example 1 for integration of the ldhL gene. After 200. mu.l of the suspension was applied to LB plates containing ampicillin (final concentration: 100. mu.g/ml) and chloramphenicol (final concentration: 34. mu.g/ml) and cultured overnight at 30 ℃, single colonies were selected and verified by PCR using primers XZ-ldhA-up/XZ-ldhA-down (consisting of XZ-ldhA-up and XZ-ldhA-down in Table 2), and the correct PCR product was 3448bp (426 bp for the upstream homology of ldhA gene, 2619bp for cat-sacB expression cassette, and 403bp for the downstream homology of ldhA gene), and a single correct colony was selected and named as Escherichia coli Slac 005.

In the second step, 476bp DNA fragment II (426 bp for the upstream homology arm of the ldhA gene and 50bp for the downstream homology arm of the ldhA gene) was amplified using the genomic DNA of E.coli Dlac-206 as a template and the primers XZ-ldhA-up/ldhA-del-down (consisting of XZ-ldhA-up and ldhA-del-down in Table 2). The DNA fragment II was used for the second homologous recombination. The DNA fragment II was electroporated into E.coli Slac 005.

The electrotransfer conditions and procedures were identical to the second procedure described in example 1 for integration of the ldhL gene. Cloning was verified by colony PCR using XZ-ldhA-up/XZ-ldhA-down as primers, a 829bp fragment (426 bp upstream homology arm of ldhA gene and 403bp downstream homology arm of ldhA gene) as a correct colony amplification product, and a correct single colony was selected and named as E.coli Slac006 (Table 1).

Compared with Escherichia coli Dlac-206, Escherichia coli Slac006 is a recombinant bacterium obtained by transforming Escherichia coli Dlac-206 as follows: the D-lactate dehydrogenase encoding gene (abbreviated as ldhA) of Escherichia coli Dlac-206 was deleted, and an L-lactate dehydrogenase gene expression cassette was introduced into the site of the fumarate reductase encoding gene (abbreviated as frd) of Escherichia coli Dlac-206. The nucleotide sequence of the L-lactate dehydrogenase gene expression cassette is sequence 2 in the sequence table. The sequence 2 in the sequence table is composed of 1051 nucleotides, the 1 st to 88 th nucleotides are M1-93 promoters, and the 89 th to 1051 th nucleotides are CDS (coding sequences) of L-lactate dehydrogenase genes.

Example 4 production of L-lactic acid Using recombinant E.coli Slac006

The seed culture medium consists of the following components (solvent is water):

macroelements: glucose 20g/L, NH₄H₂PO₄ 0.87g/L、(NH₄)₂HPO₄ 2.63g/L、MgSO₄7H2O0.18g/L, betaine-HCl 0.15 g/L.

Trace elements: FeCl₃·6H₂O 1.5μg/L、CoCl₂·6H₂O 0.1μg/L、CuCl₂·2H₂O 0.1μg/L、ZnCl₂ 0.1μg/L、Na₂MoO₄·2H₂O 0.1μg/L、MnCl₂·4H₂O 0.2μg/L,H₃BO₃ 0.05μg/L。

The fermentation medium was mostly identical to the seed medium, except that the glucose concentration was 100 g/L.

Anaerobic fermentation of the Slac006 strain, comprising the following steps:

(1) seed culture: the seed culture medium in a 250ml triangular flask is 100ml, and sterilized for 15min at 115 ℃. After cooling, the recombinant Escherichia coli Slac006 was inoculated into a seed culture medium in an inoculum size of 1% (V/V), and cultured at 37 ℃ and 100rpm for 12 hours to obtain a seed solution for inoculation of a fermentation medium.

(2) Fermentation culture: the volume of the fermentation medium in a 500ml anaerobic jar is 250ml, and the seed liquid is added according to the final concentrationOD₅₅₀The fermentation medium was inoculated with an inoculum size of 0.1, and the mixture was fermented at 37 ℃ and 150rpm for 2 days to obtain a fermentation broth. The neutralizing agent was 5M ammonia water, and the pH of the fermentor was controlled to 7.0. The fermentation liquor is all substances in the fermentation tank. No gas is introduced during the culture.

The analysis method comprises the following steps: the components in the fermentation broth were measured for 48 hours using an Agilent (Agilent-1200) high performance liquid chromatograph. The concentration of glucose and organic acid in the fermentation broth was measured using an Aminex HPX-87H organic acid analytical column from Biorad. The optical purity of lactic acid was analyzed by using a SUMICHIRAL OA-6000 chiral column from Sumika Chemical Analysis Service, Japan.

As a result, it was found (FIG. 1) that Slac006 was fermented for 48 hours at OD₅₅₀2.36, the yield of the L-lactic acid reaches 60.6g/L, the optical purity is close to 100 percent, the generation of the D-lactic acid is not detected in the chiral column, and the saccharic acid conversion rate reaches 93 percent.

Example 5 construction of recombinant E.coli Slac007

Starting from Escherichia coli Slac006, cell growth and L-lactic acid production capacity are synchronously improved through evolution and metabolism.

The fermentation medium used for the evolutionary metabolism consists of the following components (solvent is water):

macroelements: glucose 100g/L, NH₄H₂PO₄ 0.87g/L、(NH₄)₂HPO₄ 2.63g/L、MgSO₄·7H₂O0.18g/L, betaine-HCl 0.15 g/L.

Trace elements: FeCl₃·6H₂O 2.4μg/L、CoCl₂·6H₂O 0.3μg/L、CuCl₂·2H₂O 0.15μg/L、ZnCl₂ 0.3μg/L、Na₂MoO₄·2H₂O 0.3μg/L、MnCl₂·4H₂O 0.5μg/L,H₃BO₃ 0.072μg/L。

The evolutionary metabolism process used a 500mL fermenter with 250mL of fermentation medium. The pH of the fermentor was controlled at 7.0 using 5M ammonia as neutralizing agent.

No gas is introduced during the culture.

In the 1 st to 13 th generations, the fermentation temperature was 41 ℃ and the fermentation broth was transferred to a new fermentor every 24 hours to bring the initial OD550nm to 0.05.

In the 14 th to 27 th generations, the fermentation temperature was 43 ℃ and the fermentation broth was transferred to a new fermentor every 24 hours to bring the initial OD550nm to 0.05.

In 28 th to 70 th generations, the fermentation temperature was 45 ℃ and the fermentation broth was transferred to a new fermentor every 24 hours to bring the initial OD550 to 0.05.

At 71-105 th passage, the fermentation temperature was 46 ℃ and the fermentation broth was transferred to a new fermentor every 24 hours to bring the initial OD550 to 0.05.

After 105 generations of evolution, recombinant E.coli Slac007 (FIG. 2) was obtained. The recombinant Escherichia coli Slac007 is Escherichia coli (Escherichia coli), and the registration number of the recombinant Escherichia coli is CGMCC No.19459 in the China general microbiological culture Collection center (CGMCC).

Example 6 fermentative production of L-lactic acid Using recombinant E.coli Slac007 in a 500mL fermentor

The seed medium and assay were as described in example 4.

A500 mL fermentor was used, with 250mL fermentation medium. The fermentation medium was essentially the same as the seed medium except that the glucose concentration was 100g/L, the fermentation temperature was 46 ℃ and 5M ammonia was used as the neutralizing agent to control the pH of the fermentor to 7.0.

As a result, it was found (fig. 3): after Slac007 is fermented for 48 hours, the yield of L-lactic acid reaches 90g/L, the yield reaches 1.9mol/mol, the optical purity is close to 100%, and D-lactic acid is not generated in a chiral column under the condition of the invention.

Example 7 fermentative production of L-lactic acid Using recombinant E.coli Slac007 in a 5L fermentor

The L-lactic acid content and the optical purity of L-lactic acid in the seed medium and the fermentation broth were analyzed as described in example 4. The fermentation medium was largely identical to the seed medium, except that the glucose concentration was 180 g/L. The fermentation medium is obtained by replacing the glucose concentration of the seed culture medium from 20g/L to 180g/L, and keeping the other components unchanged. The pH does not need to be adjusted in the preparation process of the fermentation medium, and the system can automatically adjust the pH in the fermentation process.

Anaerobic fermentation of recombinant E.coli Slac007 in a 5L fermentor (Shanghai Baoxin, BIOTECH-5BG) comprising the following steps:

(1) seed culture: 150mL of seed culture medium in a 500mL triangular flask, and sterilizing at 115 ℃ for 15 min. After cooling, the recombinant Escherichia coli Slac007 is inoculated on a seed culture medium according to the inoculation amount of 1% (V/V), and cultured for 12 hours at 37 ℃ and 100rpm to obtain a seed solution for inoculation of a fermentation culture medium.

(2) Fermentation culture: the 5L fermenter was filled with 3L fermentation medium and sterilized at 115 ℃ for 25 min. Inoculating the seed solution into fermentation medium, allowing OD550nm of the inoculated fermentation medium to be 0.2 (using non-inoculated fermentation medium as blank control), performing anaerobic culture at 46 deg.C for 2 days, and stirring at 200rpm to obtain fermentation liquid. The fermentation liquor is all substances in the fermentation tank. The culture process is not aerated.

As a result, it was found (fig. 4): after 48 hours of fermentation, the yield of L-lactic acid reaches 150g/L, the yield reaches 1.90mol/mol, the optical purity is close to 100 percent, and D-lactic acid is not generated in the chiral column under the condition of the invention.

Example 8 genomic analysis of recombinant E.coli Slac007

The genomes of the e.coli Slac006 of example 3 and the recombinant e.coli Slac007 of example 5 were sequenced, comprising the following steps:

genomic DNAs of Escherichia coli Slac006 of example 3 and recombinant Escherichia coli Slac007 of example 5 were extracted with DNA extraction kits (available from Promega corporation, Inc.), (

Genomic DNA Purification Kit, cat # A1120), and the specific steps are completed according to the Kit instructions. The detection of DNA concentration was done quantitatively by the Qubit Fluorometer and agarose gel electrophoresis.

Genome re-sequencing was performed by norrow-derived biotechnology limited. Sequencing was performed by constructing a library of small fragments of microorganisms, with a desired data volume of 2Gb per sample. The reference sequence for sequence analysis was ATCC8739 of genomic sequence (http://www.ncbi.nlm.nih.gov/nuccore/NC_010468.1)。

It was found by genome re-sequencing analysis that the recombinant escherichia coli Slac007 of example 5 underwent 2-point mutation in the L-lactate dehydrogenase gene (ldhL) at the position of fumarate reductase encoding gene (frd) of escherichia coli Slac006 of example 3, the 133 th nucleotide was changed from a to G, the 772 th nucleotide was changed from G to a, the corresponding 45 th amino acid was changed from Threonine (Threonine) to alanine (alanine), and the 258 th amino acid was changed from Glycine (Glycine) to Serine (Serine). The mutated gene was named L-lactate dehydrogenase mutant gene (abbreviated as ldhL), and the protein encoded by ldhL was named L-lactate dehydrogenase mutant. The amino acid sequence of the L-lactate dehydrogenase mutant is a sequence 4 in a sequence table. The L-lactate dehydrogenase mutant has a modification in which T is substituted with A at position 45 and a modification in which G is substituted with S at position 258 in the amino acid sequence shown in SEQ ID NO. 4, as compared with L-lactate dehydrogenase.

Example 9 determination of L-lactate dehydrogenase Activity in recombinant E.coli Slac006 and Slac007

(1) Fermentation culture:

the seed culture and fermentation culture of Slac006 were the same as in example 4.

The seed culture and fermentation culture of Slac007 were the same as in example 6.

(2) Preparation of crude enzyme solution of cells:

the fermentation broth is cultured to logarithmic growth phase. 15ml of fermentation broth in the logarithmic phase was collected by centrifugation at 4 ℃. Washing with precooled Tris-HCl (pH7.5) for 2 times; the cells were resuspended in 1.5ml of Tris-HCl (pH 7.5). The cells were disrupted by ultrasonic cell disrupter (SCIENTZ-II0, Ningbo Xin Zhi Biotech Co., Ltd.) with ultrasonic intensity of 30% for 7min and ultrasonic process of 1sec and 2 sec. Finally, the disrupted cells were centrifuged at 12000rpm at 4 ℃ for 20min to remove cell debris.

And (3) measuring the total protein concentration of the crude enzyme liquid: the total Protein concentration of the crude enzyme solution was measured using a Bio-Rad Protein Assay kit (Berlod Biotechnology Co., Ltd.) according to the instructions provided in the kit.

(3) Activity measurement of L-lactate dehydrogenase

1mL of the reaction solution contained 100mM Tris-HCl buffer (pH8.0), 0.5mM NADH, 10mM pyruvic acid, and 10. mu.l of the crude enzyme solution. Because pyruvate can be reduced to L-lactate by NADH under the action of L-lactate dehydrogenase. Thus, the decrease in absorbance of NADH at 340nm can be measured to determine the lactate dehydrogenase enzyme activity; extinction coefficient of 6.3mM^-1cm^-1. The unit of enzyme activity is defined as: under the above conditions, the reaction is carried out for 1min, and the enzyme amount required by 1 mu mol NADH is reduced to be one enzyme activity unit. The specific activity of L-lactate dehydrogenase was calculated as the activity of the enzyme per unit total protein in U/mg.

As a result, the enzyme activity of the L-lactate dehydrogenase in the recombinant Escherichia coli Slac007 was 1.9U/mg protein, which was 1.9 times that of the L-lactate dehydrogenase in the starting strain Slac006 (the enzyme activity was 1U/mg protein).

Sequence listing

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

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Thr Ala Gly Ala Pro Gln Lys Pro Gly Glu Ser Arg Leu Asp Leu Val

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Asn Lys Asn Leu Asn Ile Leu Ser Ser Ile Val Lys Pro Val Val Asp

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Ser Gly Phe Asp Gly Ile Phe Leu Val Ala Ala Asn Pro Val Asp Ile

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Claims

1. A recombinant escherichia coli, characterized in that: the recombinant escherichia coli does not contain a D-lactate dehydrogenase gene and contains an L-lactate dehydrogenase mutant gene, the recombinant escherichia coli expresses the L-lactate dehydrogenase mutant gene, and the encoded amino acid sequence of the L-lactate dehydrogenase mutant gene is a protein of a sequence 4 in a sequence table;

the recombinant escherichia coli is obtained by modifying a receptor escherichia coli, wherein the receptor escherichia coli is escherichia coli Dlac-206, and the registration accession number of the escherichia coli Dlac-206 in the China general microbiological culture Collection center is CGMCC No. 7679;

the L-lactate dehydrogenase mutant gene is introduced into the fumaric acid reductase coding gene site of the receptor escherichia coli.

2. The recombinant E.coli of claim 1, wherein: the L-lactate dehydrogenase mutant gene is a DNA or cDNA molecule of which the coding sequence is 89-1051 site nucleotide of a sequence 3 in a sequence table;

or the like, or, alternatively,

the L-lactate dehydrogenase mutant gene is ldhL, and the encoded polypeptide contains a modification of replacing T with A at the 45 th position and a modification of replacing G with S at the 258 th position in the amino acid sequence shown in a sequence 4 in a sequence table.

3. The recombinant E.coli of claim 1 or 2, wherein: the recombinant escherichia coli contains an L-lactate dehydrogenase mutant gene expression cassette which contains a promoter and the L-lactate dehydrogenase mutant gene driven by the promoter.

4. The recombinant E.coli of claim 3, wherein: the promoter is an M1-93 promoter, and the M1-93 promoter is a DNA molecule of which the nucleotide sequence of one strand is the 1 st-88 th nucleotide of the sequence 3 in the sequence table.

5. The recombinant E.coli of claim 4, wherein: the recombinant escherichia coli is recombinant escherichia coli Slac007 which has a registration accession number of CGMCC No.19459 in the China general microbiological culture Collection center.

6. The method for constructing recombinant Escherichia coli as claimed in any one of claims 1 to 5, which comprises transforming the recipient Escherichia coli to obtain the recombinant Escherichia coli: knocking out a D-lactate dehydrogenase gene of the receptor escherichia coli, and expressing the L-lactate dehydrogenase mutant gene in the receptor escherichia coli, wherein the receptor escherichia coli is escherichia coli Dlac-206, and the registration number of the escherichia coli Dlac-206 in China general microbiological culture Collection center is CGMCC No. 7679;

7. A method for producing L-lactic acid comprising culturing the recombinant Escherichia coli of any one of claims 1 to 5 to obtain a fermentation product; obtaining L-lactic acid from the fermentation product.

8. A protein or biomaterial related thereto, characterized in that: the protein is a protein with an amino acid sequence shown as a sequence 4 in a sequence table, and the biological material is any one of the following materials:

B1) a nucleic acid molecule encoding the protein;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector.

9. Any one of the following applications:

use of P1, the recombinant escherichia coli of any one of claims 1 to 5, or the method of claim 6 for the production of L-lactic acid;

use of P2, the protein of claim 8, or a biological material related thereto for the production of L-lactic acid;

use of P3, the recombinant E.coli of any one of claims 1 to 5 or the method of claim 6 for the preparation of L-lactate dehydrogenase;

use of P4, the L-lactate dehydrogenase mutant gene described in claim 1 or 2, or the L-lactate dehydrogenase mutant gene expression cassette described in claim 3 for constructing recombinant Escherichia coli producing L-lactate dehydrogenase;

use of P5, the L-lactate dehydrogenase mutant gene according to claim 1 or 2, or the L-lactate dehydrogenase mutant gene expression cassette according to claim 3 for the preparation of L-lactate dehydrogenase;

use of P6, the protein of claim 8, or a biological material related thereto for the preparation of an L-lactate dehydrogenase preparation.